The long-term objective is to investigate the enzymes and signal transduction processes of steroid hormone biosynthesis. The current emphasis is on the mechanism by which the peptide hormone ACTH (adrenocorticotropic hormone) regulates the rate-limiting side-chain cleavage of cholesterol, an inner mitochondrial membrane reaction which is catalyzed by cytochrome P-450scc and associated enzymes. We will emphasize during the current grant period the regulation by ACTH and cAMP of cholesterol trafficking within the adrenal cell, and the regulation of this process by putative regulatory factors including Steroidogenesis Activator Polypeptide, Sterol Carrier Protein-2, nucleotide triphosphates, and cholesterol sulfate. Experimental systems to be used include the Y-1 adrenal and MA-10 Leydig cell lines. Methods to be used include cell permeabilization with Streptolysin O and generation of "semi-intact" cells. In addition, recombinant methodology and cell transfection will be used to express putative protein and peptide regulators in Y-1 cells. Subfractionation studies will also be used to localize functional cholesterol pools which are involved in moving cholesterol to its inner mitochondrial membrane metabolic site. Emphasis will be placed on a putative cholesterol-rich vesicle and inner-outer mitochondrial membrane contact junctions.